For Duchenne muscular dystrophy—the most severe and common form of a heterogeneous group of inheritable muscle wasting disorders—a solution might lie in reframing the problem. All muscular dystrophies are caused by mutations in the DMD gene, which encodes dystrophin, a large cytoplasmic protein that plays a critical role in muscle-fiber integrity. Duchenne muscular dystrophy occurs only when the DMD mutations cause reading-frame disruptions that result in nonfunctional forms of dystrophin. In contrast, in-frame deletions in the DMD gene yield internally truncated but functional dystrophins that cause a less severe form of the disease called Becker muscular dystrophy.

In a new study, Maggio and colleagues employed CRISPR/Cas9 gene editing technology to delete internal portions of the DMD gene in muscle progenitor cells from patients. By moving the DMD reading frame back on track, the authors facilitated the expression of Becker-like dystrophin proteins, which have been shown to confer therapeutic benefit in mouse models of Duchenne muscular dystrophy. An adenoviral vector was used to deliver, into patient-derived muscle progenitor cells, a plasmid that encoded the nuclease Cas9 and one or two guide RNAs (gRNAs), which are the components needed to induce gene knockouts using the CRISPR/Cas9 machinery. The adenovirus vector used in this study offered several advantages, including transient gene expression and a packaging capacity large enough to accommodate an “all-in-one” Cas9/gRNA-encoding plasmid. The DNA cargo of adenovirus does not incorporate into the genome, circumventing the cancer risk associated with traditional gene therapy and preventing unnecessary permanent expression of gene-editing components.

Experiments demonstrated the utility of delivering single gRNAs that edit a small region of the DMD gene or two gRNAs that target each end of a mutation-prone hotspot region, resulting in its complete deletion. Because 60% of patients experience mutations in the hotspot between exons 45 and 55, the latter strategy could be used, in theory, to treat a majority of Duchenne muscle dystrophy patients without the need for customization. When employing single or multiplexed gRNAs, gene editing rescued DMD activity in more than 10% of muscle cells, inducing the expression of modified or truncated (359 kDa) dystrophin. Future work is needed to assess the functionality of the rescued dystrophin in a Duchenne muscular dystrophy mouse model, as well as the effects of the adenovirus delivery vehicle on the human immune system.